Multi-molecular hyperspectral PRM-SRS microscopy.

Lipids play crucial roles in many biological processes. Mapping spatial distributions and examining the metabolic dynamics of different lipid subtypes in cells and tissues are critical to better understanding their roles in aging and diseases. Commonly used imaging methods (such as mass spectrometry-based, fluorescence labeling, conventional optical imaging) can disrupt the native environment of cells/tissues, have limited spatial or spectral resolution, or cannot distinguish different lipid subtypes. Here we present a hyperspectral imaging platform that integrates a Penalized Reference Matching algorithm with Stimulated Raman Scattering (PRM-SRS) microscopy. Using this platform, we visualize and identify high density lipoprotein particles in human kidney, a high cholesterol to phosphatidylethanolamine ratio inside granule cells of mouse hippocampus, and subcellular distributions of sphingosine and cardiolipin in human brain. Our PRM-SRS displays unique advantages of enhanced chemical specificity, subcellular resolution, and fast data processing in distinguishing lipid subtypes in different organs and species.

Integration of FUNDC1-associated mitochondrial protein import and mitochondrial quality control contributes to TDP-43 degradation.

Though TDP-43 protein can be translocated into mitochondria and causes mitochondrial damage in TDP-43 proteinopathy, little is known about how TDP-43 is imported into mitochondria. In addition, whether mitochondrial damage is caused by mitochondrial mislocalization of TDP-43 or a side effect of mitochondria-mediated TDP-43 degradation remains to be investigated. Here, our bioinformatical analyses reveal that mitophagy receptor gene FUNDC1 is co-expressed with TDP-43, and both TDP-43 and FUNDC1 expression is correlated with genes associated with mitochondrial protein import pathway in brain samples of patients diagnosed with TDP-43 proteinopathy. FUNDC1 promotes mitochondrial translocation of TDP-43 possibly by promoting TDP-43-TOM70 and DNAJA2-TOM70 interactions, which is independent of the LC3 interacting region of FUNDC1 in cellular experiments. In the transgenic fly model of TDP-43 proteinopathy, overexpressing FUNDC1 enhances TDP-43 induced mitochondrial damage, whereas down-regulating FUNDC1 reverses TDP-43 induced mitochondrial damage. FUNDC1 regulates mitochondria-mediated TDP-43 degradation not only by regulating mitochondrial TDP-43 import, but also by increasing LONP1 level and by activating mitophagy, which plays important roles in cytosolic TDP-43 clearance. Together, this study not only uncovers the mechanism of mitochondrial TDP-43 import, but also unravels the active role played by mitochondria in regulating TDP-43 homeostasis.

Bioorthogonal Stimulated Raman Scattering Imaging Uncovers Lipid Metabolic Dynamics in Drosophila Brain During Aging.

Studies have shown that brain lipid metabolism is associated with biological aging and influenced by dietary and genetic manipulations; however, the underlying mechanisms are elusive. High-resolution imaging techniques propose a novel and potent approach to understanding lipid metabolic dynamics in situ. Applying deuterium water (D2O) probing with stimulated Raman scattering (DO-SRS) microscopy, we revealed that lipid metabolic activity in Drosophila brain decreased with aging in a sex-dependent manner. Female flies showed an earlier occurrence of lipid turnover decrease than males. Dietary restriction (DR) and downregulation of insulin/IGF-1 signaling (IIS) pathway, two scenarios for lifespan extension, led to significant enhancements of brain lipid turnover in old flies. Combining SRS imaging with deuterated bioorthogonal probes (deuterated glucose and deuterated acetate), we discovered that, under DR treatment and downregulation of IIS pathway, brain metabolism shifted to use acetate as a major carbon source for lipid synthesis. For the first time, our study directly visualizes and quantifies spatiotemporal alterations of lipid turnover in Drosophila brain at the single organelle (lipid droplet) level. Our study not only demonstrates a new approach for studying brain lipid metabolic activity in situ but also illuminates the interconnection of aging, dietary, and genetic manipulations on brain lipid metabolic regulation.

Neuronal guidance genes in health and diseases.

Neurons migrate from their birthplaces to the destinations, and extending axons navigate to their synaptic targets by sensing various extracellular cues in spatiotemporally controlled manners. These evolutionally conserved guidance cues and their receptors regulate multiple aspects of neural development to establish the highly complex nervous system by mediating both short- and long-range cell-cell communications. Neuronal guidance genes (encoding cues, receptors, or downstream signal transducers) are critical not only for development of the nervous system but also for synaptic maintenance, remodeling, and function in the adult brain. One emerging theme is the combinatorial and complementary functions of relatively limited classes of neuronal guidance genes in multiple processes, including neuronal migration, axonal guidance, synaptogenesis, and circuit formation. Importantly, neuronal guidance genes also regulate cell migration and cell-cell communications outside the nervous system. We are just beginning to understand how cells integrate multiple guidance and adhesion signaling inputs to determine overall cellular/subcellular behavior and how aberrant guidance signaling in various cell types contributes to diverse human diseases, ranging from developmental, neuropsychiatric, and neurodegenerative disorders to cancer metastasis. We review classic studies and recent advances in understanding signaling mechanisms of the guidance genes as well as their roles in human diseases. Furthermore, we discuss the remaining challenges and therapeutic potentials of modulating neuronal guidance pathways in neural repair.

Super-resolution SRS microscopy with A-PoD.

Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (D2O)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.

Detection of orthologous exons and isoforms using EGIO.

MOTIVATION: Alternative splicing is an important mechanism to generate transcriptomic and phenotypic diversity. Existing methods have limited power to detect orthologous isoforms. RESULTS: We develop a new method, EGIO, to detect orthologous exons and orthologous isoforms from two species. EGIO uses unique exonic regions to construct exon groups, in which process dynamic programming strategy is used to do exon alignment. EGIO could cover all the coding exons within orthologous genes. A comparison between EGIO and ExTraMapper shows that EGIO could detect more orthologous isoforms with conserved sequence and exon structures. We apply EGIO to compare human and chimpanzee protein-coding isoforms expressed in the frontal cortex and identify 6912 genes that express human unique isoforms. Unexpectedly, more human unique isoforms are detected than those conserved between humans and chimpanzees. AVAILABILITY AND IMPLEMENTATION: Source code and test data of EGIO are available at https://github.com/wu-lab-egio/EGIO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking.

Tunneling nanotubes (TNTs) are actin-rich structures that connect two or more cells and mediate cargo exchange between spatially separated cells. TNTs transport signaling molecules, vesicles, organelles, and even pathogens. However, the molecular mechanisms regulating TNT formation remain unclear and little is known about the endogenous mechanisms suppressing TNT formation in lung cancer cells. Here, we report that MICAL2PV, a splicing isoform of the neuronal guidance gene MICAL2, is a novel TNT regulator that suppresses TNT formation and modulates mitochondrial distribution. MICAL2PV interacts with mitochondrial Rho GTPase Miro2 and regulates subcellular mitochondrial trafficking. Moreover, down-regulation of MICAL2PV enhances survival of cells treated with chemotherapeutical drugs. The monooxygenase (MO) domain of MICAL2PV is required for its activity to inhibit TNT formation by depolymerizing F-actin. Our data demonstrate a previously unrecognized function of MICAL2 in TNT formation and mitochondrial trafficking. Furthermore, our study uncovers a role of the MICAL2PV-Miro2 axis in mitochondrial trafficking, providing a mechanistic explanation for MICAL2PV activity in suppressing TNT formation and in modulating mitochondrial subcellular distribution.

Direct Imaging of Lipid Metabolic Changes in Drosophila Ovary During Aging Using DO-SRS Microscopy.

Emerging studies have shown that lipids and proteins play versatile roles in various aspects of aging. High-resolution in situ optical imaging provides a powerful approach to study the metabolic dynamics of lipids and proteins during aging. Here, we integrated D2O probing and stimulated Raman scattering (DO-SRS) microscopy to directly visualize metabolic changes in aging Drosophila ovary. The subcellular spatial distribution of de novo protein synthesis and lipogenesis in ovary was quantitatively imaged and examined. Our Raman spectra showed that early stages follicles were protein-enriched whereas mature eggs were lipid-enriched. DO-SRS imaging showed a higher protein synthesis in the earlier developing stages and an increased lipid turned over at the late stage. Aged (35 days) flies exhibited a dramatic decrease in metabolic turnover activities of both proteins and lipids, particularly, in the germ stem cell niche of germarium. We found an accumulation of unsaturated lipids in the nurse cells and oocytes in old flies, suggesting that unsaturated lipids may play an important role in the processes of oocyte maturation. We further detected changes in mitochondrial morphology and accumulation of Cytochrome c during aging. To our knowledge, this is the first study that directly visualizes spatiotemporal changes in lipid and protein metabolism in Drosophila ovary during development and aging processes. Our study not only demonstrates the application of a new imaging platform in visualizing metabolic dynamics of lipids and proteins in situ but also unravels how the metabolic activity and lipid distribution change in Drosophila ovary during aging.

SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling.

Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa.

TDP-43 induces mitochondrial damage and activates the mitochondrial unfolded protein response.

Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPRmt) in both cellular and animal models. Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases.

Reduced USP33 expression in gastric cancer decreases inhibitory effects of Slit2-Robo1 signalling on cell migration and EMT.

OBJECTIVES: Gastric cancer (GC) is one of the most common cancers in the world, causing a large number of deaths every year. The Slit-Robo signalling pathway, initially discovered for its critical role in neuronal guidance, has recently been shown to modulate tumour invasion and metastasis in several human cancers. However, the role of Slit-Robo signalling and the molecular mechanisms underlying its role in the pathogenesis of gastric cancer remains to be elucidated. MATERIALS AND METHODS: Slit2, Robo1 and USP33 expressions were analysed in datasets obtained from the Oncomine database and measured in human gastric cancer specimens. The function of Slit2-Robo1-USP33 signalling on gastric cancer cells migration and epithelial-mesenchymal transition (EMT) was studied both in vitro and in vivo. The mechanism of the interaction between Robo1 and USP33 was explored by co-IP and ubiquitination protein analysis. RESULTS: The mRNA and protein levels of Slit2 and Robo1 are lower in GC tissues relative to those in adjacent healthy tissues. Importantly, Slit2 inhibits GC cell migration and suppresses EMT process in a Robo-dependent manner. The inhibitory function of Slit2-Robo1 is mediated by ubiquitin-specific protease 33 (USP33) via deubiquitinating and stabilizing Robo1. USP33 expression is decreased in GC tissues, and reduced USP33 level is correlated with poor patient survival. CONCLUSIONS: Our study reveals the inhibitory function of Slit-Robo signalling in GC and uncovers a role of USP33 in suppressing cancer cell migration and EMT by enhancing Slit2-Robo1 signalling. USP33 represents a feasible choice as a prognostic biomarker for GC.

A crucial role for Arf6 in the response of commissural axons to Slit.

A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.

Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation.

Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an "open-book" spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and "closed-book" spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.

FUS interacts with ATP synthase beta subunit and induces mitochondrial unfolded protein response in cellular and animal models.

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPRmt). Importantly, down-regulating expression of ATP5B or UPRmt genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration.

Measure and model a 3-D space-variant PSF for fluorescence microscopy image deblurring.

Conventional deconvolution methods assume that the microscopy system is spatially invariant, introducing considerable errors. We developed a method to more precisely estimate space-variant point-spread functions from sparse measurements. To this end, a space-variant version of deblurring algorithm was developed and combined with a total-variation regularization. Validation with both simulation and real data showed that our PSF model is more accurate than the piecewise-invariant model and the blending model. Comparing with the orthogonal basis decomposition based PSF model, our proposed model also performed with a considerable improvement. We also evaluated the proposed deblurring algorithm. Our new deblurring algorithm showed a significantly better signal-to-noise ratio and higher image quality than those of the conventional space-invariant algorithm.

Traumatic injury induces stress granule formation and enhances motor dysfunctions in ALS/FTD models.

Traumatic brain injury (TBI) has been predicted to be a predisposing factor for amyotrophic lateral sclerosis (ALS) and other neurological disorders. Despite the importance of TBI in ALS progression, the underlying cellular and molecular mechanisms are still an enigma. Here, we examined the contribution of TBI as an extrinsic factor and investigated whether TBI influences the susceptibility of developing neurodegenerative symptoms. To evaluate the effects of TBI in vivo, we applied mild to severe trauma to Drosophila and found that TBI leads to the induction of stress granules (SGs) in the brain. The degree of SGs induction directly correlates with the level of trauma. Furthermore, we observed that the level of mortality is directly proportional to the number of traumatic hits. Interestingly, trauma-induced SGs are ubiquitin, p62 and TDP-43 positive, and persistently remain over time suggesting that SGs might be aggregates and exert toxicity in our fly models. Intriguingly, TBI on animals expressing ALS-linked genes increased mortality and locomotion dysfunction suggesting that mild trauma might aggravate neurodegenerative symptoms associated with ALS. Furthermore, we found elevated levels of high molecular weight ubiquitinated proteins and p62 in animals expressing ALS-causing genes with TBI, suggesting that TBI may lead to the defects in protein degradation pathways. Finally, we observed that genetic and pharmacological induction of autophagy enhanced the clearance of SGs and promoted survival of flies in vivo. Together, our study demonstrates that trauma can induce SG formation in vivo and might enhance neurodegenerative phenotypes in the fly models of ALS.

TDP-43 regulates cancer-associated microRNAs.

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.

Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies.

Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria. Crude mitochondria of rabbit liver obtained from differential centrifugation were purified by the developed method according to their pI values as six fractions. Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61, degenerative mitochondria were in two fractions of pH 5.46 and 5.72, and inner membrane vesicles (IMVs) appeared in the fractions of pH 4.70 and 5.04. Membrane potential measurement proved a dramatic difference between intact mitochondria and IMVs, which reflected the bioactivity of obtained populations. Particularly, proteomics analyses revealed that more number of proteins were identified in the intact fractions than that of IMVs or crude mitochondria, which demonstrated that RFFIEF could be powerful tool for the preparation of intact organelle as well as their proteomic and in-depth biological analysis.

PINK1 and Parkin are genetic modifiers for FUS-induced neurodegeneration.

Dysregulation of Fused in Sarcoma (FUS) gene expression is associated with fronto-temporal lobar degeneration (FTLD), and missense mutations in the FUS gene have been identified in patients affected by amyotrophic lateral sclerosis (ALS). However, molecular and cellular defects underlying FUS proteinopathy remain to be elucidated. Here, we examined whether genes important for mitochondrial quality control play a role in FUS proteinopathy. In our genetic screening, Pink1 and Park genes were identified as modifiers of neurodegeneration phenotypes induced by wild type (Wt) or ALS-associated P525L-mutant human FUS. Down-regulating expression of either Pink1 or Parkin genes ameliorated FUS-induced neurodegeneration phenotypes. The protein levels of PINK1 and Parkin were elevated in cells overexpressing FUS. Remarkably, ubiquitinylation of Miro1 protein, a downstream target of the E3 ligase activity of Parkin, was also increased in cells overexpressing FUS protein. In fly motor neurons expressing FUS, both motility and processivity of mitochondrial axonal transport were reduced by expression of either Wt- or P525L-mutant FUS. Finally, down-regulating PINK1 or Parkin partially rescued the locomotive defects and enhanced the survival rate in transgenic flies expressing FUS. Our data indicate that PINK1 and Parkin play an important role in FUS-induced neurodegeneration. This study has uncovered a previously unknown link between FUS proteinopathy and PINK1/Parkin genes, providing new insights into the pathogenesis of FUS proteinopathy.